Download Glutamate 83 and arginine 85 of helix H3 bend are key residues for

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RESEARCH ARTICLE
Open Access
Glutamate 83 and arginine 85 of helix H3 bend
are key residues for FtsZ polymerization, GTPase
activity and cellular viability of Escherichia coli:
lateral mutations affect FtsZ polymerization and
E. coli viability
Jae Yen Shin1, Waldemar Vollmer2, Rosalba Lagos3 and Octavio Monasterio3*
Abstract
Background: FtsZ is an essential cell division protein, which localizes at the middle of the bacterial cell to mediate
cytokinesis. In vitro, FtsZ polymerizes and induces GTPase activity through longitudinal interactions to form the
protofilaments, whilst lateral interactions result within formation of bundles. The interactions that participate in the
protofilaments are similar to its eukaryotic homologue tubulin and are well characterized; however, lateral
interactions between the inter protofilaments are less defined. FtsZ forms double protofilaments in vitro, though the
key elements on the interface of the inter-protofilaments remain unclear as well as the structures involved in the
lateral interactions in vivo and in vitro. In this study, we demonstrate that the highly conserved negative charge of
glutamate 83 and the positive charge of arginine 85 located in the helix H3 bend of FtsZ are required for in vitro
FtsZ lateral and longitudinal interactions, respectively and for in vivo cell division.
Results: The effect of mutation on the widely conserved glutamate-83 and arginine-85 residues located in the helix H3
(present in most of the tubulin family) was evaluated by in vitro and in situ experiments. The morphology of the cells
expressing Escherichia coli FtsZ (E83Q) mutant at 42°C formed filamented cells while those expressing FtsZ(R85Q)
formed shorter filamented cells. In situ immunofluorescence experiments showed that the FtsZ(E83Q) mutant formed
rings within the filamented cells whereas those formed by the FtsZ(R85Q) mutant were less defined. The expression of
the mutant proteins diminished cell viability as follows: wild type > E83Q > R85Q. In vitro, both, R85Q and E83Q
reduced the rate of FtsZ polymerization (WT > E83Q >> R85Q) and GTPase activity (WT > E83Q >> R85Q). R85Q
protein polymerized into shorter filaments compared to WT and E83Q, with a GTPase lag period that was inversely
proportional to the protein concentration. In the presence of ZipA, R85Q GTPase activity increased two fold, but no
bundles were formed suggesting that lateral interactions were affected.
Conclusions: We found that glutamate 83 and arginine 85 located in the bend of helix H3 at the lateral face are
required for the protofilament lateral interaction and also affects the inter-protofilament lateral interactions that
ultimately play a role in the functional localization of the FtsZ ring at the cell division site.
Keywords: Bacterial division, FtsZ polymerization, Longitudinal/lateral interactions, ZipA
* Correspondence: [email protected]
3
Departamento de Biología, Facultad de Ciencias, Casilla 653, Santiago, Chile
Full list of author information is available at the end of the article
© 2013 Shin et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly cited.
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Background
Bacterial cell division occurs in the middle of the dividing
cell mediated by protein complex division machinery
called divisome. FtsZ (Fts, Filamentous temperature sensitive) is the principal protein of the divisome in Escherichia
coli, and assembles into the Z ring at the cell division site;
it is bound to the inner membrane and is stabilized by
FtsA and ZipA [1,2]. This event is necessary for the
sequential addition of at least twelve other membrane
proteins responsible for peptidoglycan assembly and
membrane constriction [3]. Inactivation of the corresponding genes blocks the cell division and give rise
to long multinucleated cells that fail to propagate and
eventually die [4].
The Z-ring and its dynamics during the cell process
have been observed by fluorescence microscopy in living
cells [5] and in liposomes [6]. Although, the organization
of FtsZ in the Z-ring is not known, based on the in vitro
studies it is believed that the basic units to form the ring
are the protofilaments. In order to explain the Z-ring dynamics at the molecular level, two theoretical models have
been proposed. One model involves the hydrolysis of GTP
to generate the force for invagination of the plasma membrane [7] and the other model proposes a microphase
transition of the FtsZ filaments that is energized by lateral
interactions between filaments during the constriction of
the plasma membrane [8]. In both models, the lateral
interactions between the protofilaments are crucial for the
membrane constriction. However, the role of lateral interactions has been questioned because the interaction
entropy was not considered in the formation of the filaments [9]. Therefore, the question regarding the role of
lateral interactions still remains unclear.
Mutations on the lateral interface of FtsZ induce the
formation of an aberrant Z-ring in E. coli cells and cell
division can be compromised [10,11]. Apparently, the lateral interactions to form the Z-ring are also important in
B. subtilis [12]. Recently, the Z-ring was observed in vivo
at a higher resolution. The authors proposed that FtsZ
polymerizes into short filaments that assemble into loose
bundles mediated by longitudinal and lateral interactions
to finally locate as a Z-ring at the division site [13]. These
observations suggest that lateral interactions between FtsZ
protofilaments could be important for in vivo formation
of the functional Z ring. However, the elements that play a
key role in the Z-ring formation remain unknown.
In vitro, lateral interactions of FtsZ stabilize sheets
and rings [14] and ZipA, mentioned earlier as a membrane anchor protein, stimulates the formation of FtsZ
sheets [15]. Moreover, FtsZ also polymerizes into sheets
and tubules in the presence of cations such as calcium
or DEAE-dextran [15-18], or by the FtsZ-interacting
protein SepF [19,20]. The sheets and tubules are stabilized by lateral interactions similar to those found in
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tubulin polymers [14,21]. Moreover, arc-like filaments
that could interact laterally have been observed by electron
cryotomography at the constriction site [22], but the importance of these lateral interactions has remained controversial [23]. Ca2+-induced FtsZ sheets are composed of
protofilaments that are aligned in parallel and antiparallel
way, while in Zn2+-induced tubulin sheets are arranged
only in an antiparallel manner. Thus, from a low-resolution
sheet structure of Methanococcus jannaschii FtsZ, it has
been proposed that two protofilaments interact through the
same lateral face, in a parallel form, to produce doublestranded filaments called thick filaments, which subsequently form sheets through antiparallel interactions [15].
E. coli FtsZ also polymerases into thick filaments [24], indicating that these filaments can be the basic units to form
sheets.
Phylogenetic and structural studies show that FtsZ is
the most-conserved cell division protein and it shares
structural and functional features with eukaryotic tubulins
[25,26]. The heterodimer αβ-tubulin polymerizes into
microtubules and two types of interactions are involved
between the heterodimer subunits: the longitudinal interactions between heterodimers to form the protofilaments,
and the lateral interactions between 13 or 14 parallel
protofilaments to organize into the microtubule. The lateral interactions result from the contacts between α- and
β-tubulin subunits of neighboring protofilaments and are
mediated by central structural elements, such as the interaction of the M loop of one subunit with the H3 helix of
the neighboring subunit [27]. A newly discovered bacterial
tubulin, BtubA/B, also contains the H3 helix and M loop,
however it is believed that the last one is a reminiscent
structure from eukaryotic tubulin [28]. Interestingly, the
H3 helix is highly conserved among FtsZs while the M
loop is absent. Therefore, we believe that H3 helix of FtsZ
can participate in the lateral interactions in a similar manner to its related tubulins.
The aim of this work was to investigate the role of the
H3 helix in lateral interactions of FtsZ from E. coli. We
mutated the highly conserved glutamate 83 and arginine
85 residues to glutamine by site-directed mutagenesis
and evaluated the effect of these mutations on the viability of the cells, the cell morphology and localization of
FtsZ, as well as the GTPase activity and the ability to
polymerize in vitro, in the presence and in the absence
of calcium and ZipA.
Results
Design of FtsZ(R85Q) and FtsZ(E83Q) mutants
We choose as candidates for mutation two conserved
residues located in the lateral face of FtsZ, specifically in
the bend of H3 helix. This was based on the similarities
in the crystal structures between FtsZ and tubulin and
the degree of conservation of amino acids among FtsZ
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sequences. The three dimensional structure of an E. coli
FtsZ modela (Figure 1A) was compared with the structure of the αβ-tubulin heterodimer. The helix H3 of
tubulin, located on the lateral face and known for the
role in the lateral interactions, is also present in FtsZ.
The helix H3 of FtsZ is located in the left lateral face
(left, right, top and bottom face are the nomenclature
defined by analogy between FtsZ protofilaments and
tubulin microtubules) [27]. Helix H3 is present in most
of the FtsZ sequences from gram positive and negative
bacteria, mitochondria and chloroplasts. Within the H3
helix, glutamate at position 83 is 100% conserved and the
positive charge of arginine at 85 is highly conserved (80%
using non-redundant sequences) among FtsZ sequences
from 11 different organisms (Figure 1B). Interestingly, the
two residues are located in the bend of the helix H3 and
are exposed to the surface of the left face. Hence, we
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hypothesized that these residues, E83 and R85, participate
in the interactions of FtsZ to form filaments, tubules or
sheets. To this end, the bend in H3 could be necessary
to maintain both the topography of the monomer for
the interactions and the GTPase activity in the polymer.
In microtubules, the lateral interactions are principally
mediated between the helix H3 from a protofilament and
the M loop of the adjacent protofilament [29]. The M loop
is even present in bacterial tubulin A/B, though smaller
than its counterpart tubulin, and is also believed to participate in the lateral interactions to form a bacterial
microtubules (bMTs) composed by five protofilaments
[30]. However, FtsZ lacks the M loop hence, the lateral
interactions are expected to be weaker between FtsZ protofilaments [13], hence an effect on longitudinal interactions should affect both the length and the GTPase
activity of the polymers.
Figure 1 (A) Front view of the E. coli FtsZ structural model. N-and C-terminal domains are represented in brown and gray, respectively.
Arrows indicate longitudinal and lateral polymerization axes and the face of FtsZ is described as top, bottom, right and left according to the
orientation of GTP (yellow). Residues E83 and R85 locate in the bend within the H3 helix. (B) Primary sequence alignment of the H3 region of
FtsZ. The most conserved positions are colored in dark blue. The secondary structure of FtsZ is shown in the last line: alpha helix, orange
rectangle; beta strand, blue arrow; coil, grey. The arrows indicate residues E83 and R85 of the E. coli FtsZ sequence.
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Viability of E. coli VIP2(DE3) cells in the presence of FtsZ
(E83Q) and FtsZ(R85Q)
To evaluate the functionality of the FtsZ mutants in vivo,
the E83Q, and R85Q proteins were expressed independently and the cell viability was determined in a strain
containing ftsZ thermonull mutant, VIP2(DE3)/pLAR9.
The parental strain, VIP2/pLAR9 has been successfully
employed to demonstrate that FtsZ is essential for cell division and survival in E. coli [31]. For the purpose of our
study we modified the parental strain to create a DE3 lysogen of VIP2, that contains a copy of T7 RNA polymerase
gene under the control of PlacUV5, so the expression of a
target protein is inducible by IPTG addition. Thus, if VIP2
(DE3)/pLAR9 is transformed with a second plasmid that
contains the FtsZ mutants (pMFV-derivatives: pMFV-wt,
pMFV-rq and pMFV-eq, see Methods), the transformed
cells will contain a ftsZ mutant allele from pMFV-derived
plasmid under the control of a T7 promoter, a wild-type
allele encoded on plasmid pLAR9 (a temperature sensitive
replication plasmid) and a genomic null allele. In summary,
at the permissive temperature (30°C), VIP2(DE3)/pLAR9/
pMFV- will express both ftsZ alleles (from pLAR9 and
pMFV-derivatives), whereas at the restrictive temperature
(42°C) pLAR9 cannot replicate, so the ftsZ expression
originates from the pMFV-derivatives. The production
of FtsZ ceases completely after 2–3 h of shifting the
temperature, once pLAR9 is segregated by lack of replication. In this regard, this system is different from those utilizing a FtsZ mutant nonfunctional at 42°C, because in this
case after shifting to the non-permissive temperature FtsZ
will be nonfunctional almost instantly.
To simplify the nomenclature we will refer henceforth
to the strain VIP2(DE3), containing pLAR9, as VIP5.
VIP5/pMFV-wt cell grown at 42°C (positive control)
and at 30°C show similar cfu/ml in the absence of IPTG,
indicating that the basal non-induced expression level
from pMFV-wt (a high copy number plasmid) was
enough to complement the lack of FtsZ from pLAR9 at
42°C (Figure 2). Hence, in order to avoid an excessive
expression of FtsZ, IPTG was not added in all our
experiments. As expected, the survival of VIP5 at 42°C
(negative control) decreases about four orders of magnitude. The small survival fraction observed in VIP5 is
explained as due to the reversion frequency of the
chromosomal mutation [31].
Figure 2 shows that the viability of cells expressing
EcFtsZ mutants E83Q and R85Q at the restrictive
temperature diminished about 1.5 and 3 orders of magnitude, respectively, compared to the non-restrictive
temperature. These values indicate that the cells could
not support normal cell division but at the same time
the EcFtsZ mutants did not completely lose functionality in vivo. Hence, the survival assay was useful to
characterize the functionality of EcFtsZ mutants in vivo
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and the quantification of the viability of the cells expressing EcFtsZ mutants was used as an indication of the efficiency in the cell division. Consequently, the efficiency of
the different forms of EcFtsZ in terms of cell viability can
be interpreted as: wild type > E83Q > R85Q.
Cell morphology and in situ localization of FtsZ mutants
using immunofluorescence microscopy
The FtsZ ring is formed in the middle of the cell and is
attached to the cytoplasmic membrane through ZipA
[2]. In order to evaluate the effect of these mutations on
cell morphology and FtsZ localization, VIP5 cells in exponential phase of growth carrying plasmids with FtsZ
(WT), FtsZ(E83Q), and FtsZ(R85Q) mutants were incubated in the absence of IPTG at 30°C (control) and at
42°C. Figure 3 shows that, in the negative control at
42°C (pLAR9 is lost at this temperature) the cell division
is blocked, and FtsZ localization is dispersed along the
very long filamentous cells (a’), while at 30°C the cells
are normal. Cells carrying the plasmid FtsZ(WT) at 42°C
(b’) reverts the cell filamentation, and at 30°C (b) as
expected the morphology and FtsZ localization is normal. The co-expression at 30°C of FtsZ(WT) from pLAR9
with the mutants FtsZ(E83Q) (c) and FtsZ(R85Q) (d) also
display a normal phenotype. However, the expression of
the FtsZ mutants at 42°C (in the absence of FtsZ(WT))
have an effect on cell division, producing filamentous cells
(c’ and d’). Interestingly, FtsZ(E83Q) still localizes as
discrete bands along the filamentous cells, probably at the
potential cell division sites (c’). In contrast, the expression
of FtsZ(R85Q) produced shorter filamentous cells with a
disperse localization of the protein along the filaments,
and no clear bands were observed (d’). The same experiments were performed with IPTG 0.2 mM and similar
results were obtained (not shown). In order to keep the
cells in exponential phase of growth for a longer time, the
morphology experiments were repeated starting the culture for the temperature shift at an OD600 of 0.06. After
3 h of incubation no difference in the cell morphology
of the mutants respect to the experiments presented in
Figure 3 was observed.
Localization of the FtsZ mutants in the single cell
population (not filaments) that appear at early stationary
phase (Additional file 1: Figure S1) shows that FtsZ
(E83Q) is located with greater intensity at the middle of
the cell but presents some degree of dispersion, while
FtsZ(R85Q) is rather distributed in a gradient shape
from the mid-cell toward the cell poles. The ZipA distribution was similar to that observed for FtsZ mutants
(Additional file 1: Figure S2), which suggests that the
mutations in FtsZ do not affect its interaction with
ZipA. Binding experiments between FtsZ (WT and
mutants) and ZipA inserted into proteoliposomes confirmed this hypothesis (not shown).
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Figure 2 Viability of E. coli VIP2 cells expressing FtsZ mutants. Exponentially-grown cells of E. coli VIP5 without (−) or with plasmids that
express wild-type FtsZ (WT), FtsZ(E83Q) or FtsZ(R85Q) were plated by duplicate on two LB agar plates and incubated at 42°C or 30°C. Cell viability
was determined the following day. The average and the standard deviation (SD) of 3 independent experiments were obtained using the
program EXCEL. The H3 bend mutations cause significant decrease in cell viability.
In summary, the residues glutamate 83 and arginine 85
located in bend of helix H3 on the left face of FtsZ plays a
role in the formation of the Z-ring (Figure 3) and consequently ZipA presents an altered localization in cells
expressing the FtsZ mutants (Additional file 1: Figure S2).
Structural characterization of R85Q and E83Q
In order to evaluate the impact of the point mutations
E83Q and R85Q on the protein structure, we performed
circular dichroism on purified FtsZ wt, R85Q and E83Q.
All three purified proteins showed the expected molecular
mass and had a very similar secondary structure content
(not shown); therefore, presumably the mutations did not
cause significant changes in the tertiary structure of FtsZ.
GTPase activity of E83Q and R85Q
In order to understand the possible effects of the FtsZ
mutations on the formation of the Z ring, we purified the
mutant proteins and measured their abilities to hydrolyze
GTP. The wild-type FtsZ and E83Q exhibited a constant
initial velocity of GTP hydrolysis for at least 10 minutes at
a protein concentration of 12.5 μM (Figure 4A). By contrast, R85Q showed a lag time (τ) of 86.3 minutes and a
reduced GTPase activity. We next compared the GTP hydrolysis efficiency by calculating the apparent catalytic
constants (i.e., the ratio between the initial velocity and
the protein concentration). Wild-type FtsZ, E83Q and
R85Q hydrolyzed GTP with apparent catalytic constants of
2.90 ± 0.16 min-1, 1.13 ± 0.06 min-1 and 0.13 ± 0.02 min-1
respectively. R85Q was further characterized by measuring
the dependence of GTP hydrolysis on protein concentration (Figure 4B). The progress curves show a sigmoidal behaviour, i.e., the lag time was dependent on the protein
concentration (inset Figure 4B). This lag time should disappear at the extrapolated concentration of 130 μM. The
similarity of the progress curves at 36.8 and 49.0 μM of
protein is probably due to GTP becoming limiting. These
results suggest that the R85Q mutation does not directly
alter the rate of GTP hydrolysis, but rather affects FtsZ
polymerization, which is known to induce GTPase activity.
Therefore, the effect is reversed when the concentration of
R85Q is increased to stimulate polymerization.
The GTPase activity of FtsZ wt and E83Q is not
affected by ZipA, which is in agreement with previous
study [2]. Interestingly, ZipA increased around twofold
the GTPase activity of R85Q (Figure 4C and Additional
file 1: Figure S3b), suggesting that ZipA stimulates the
longitudinal interactions of R85Q protofilaments during
polymerization, similar to the effect of MAPs on microtubules [32].
Polymerization of FtsZ mutants with and without ZipA
In vitro, FtsZ polymerizes into protofilaments in the
presence of GTP [33], and these protofilaments associate
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Figure 3 Cell morphology and localization of FtsZ wild type and mutants E83Q and R85Q in cells with (30°C) and without (42°C)
co-expression of FtsZ(WT) from pLAR9. Immunofluorescence of FtsZ (green) overlaid with the bright field image (gray) shows the localization
of FtsZ in VIP5 cells (for strain information see Methods). Scale Bar, 5μm. Immunofluorescence was performed as described in Methods. Briefly,
cells were grown until OD600 reached 0.5, cultures were split in half and incubated at 30°C and 42°C respectively for 4 h. Cells were harvested,
fixed and immunofluorescent labeled with anti-FtsZ as first antibody and the second antibody conjugated with Alexa 488.
to form bundles in the presence of ZipA [15]. Single
protofilaments are monomers that interact longitudinally whereas double protofilaments (thick filaments)
consist of two parallel protofilaments interacting laterally [24]. Antiparallel lateral interactions of these
double filaments induce the formation of sheets or
bundles [34]. In order to evaluate the effect of the
mutations on the polymerization capability, we followed the polymerization of FtsZ mutants by 90° light
scattering at 350 nm and the polymers morphology by
electron microscopy.
The polymerization reaction was started with the
addition of the protein, and the reaction was followed
for one hour for wild type and E83Q, while R85Q was
followed for almost three hours due to the long lag
period observed for the GTPase activity (Figure 4B). The
mutant R85Q apparently did not form long filaments at
the protein concentration used for FtsZ wild type and
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Figure 4 GTPase activity of the FtsZ mutants in the presence and absence of ZipA. (A) Progress curves for the GTPase activity in a solution
containing 50 mM Mes pH 6.5, 50 mM KCl, 10 mM MgCl2 with 12.5 μM of wild type FtsZ (O), E83Q (∇ ) or R85Q (□) at 30°C. The reaction was
started by the addition of GTP at 1 mM final concentration, and GDP was determined by HPLC, as described in Material and Methods. (B)
Progress curves for the GTPase activity of R85Q. The hydrolysis of GTP was measured with the following R85Q concentrations: (▼ ) 6.2; (● ) 12.4;
(∇ ) 18.6; (O) 24.8; (∎) 31.0 and 49.6 μM ( ). The lag time (τ) was determined as the abscissa value where the line of the steepest slope of the
progress curves intersects the x-axis. The inset shows the dependence of τ on the inverse of protein concentration. The values of picomoles
indicated in the ordinate axe correspond to the amount of GDP in 100 μL of the reaction mixture (as indicated in Methods). (C) GTPase activity of
12.5 μM wild type FtsZ, E83Q and R85Q in the presence of 12 μM ZipA; the control with ZipA alone showed no GTPase activity (not shown).
Inorganic phosphate was quantified using malachite green as described in Methods and the values of the ordinate axe are multiplied by 20.
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E83Q (Figure 5C), producing a small increment in turbidity that was stimulated around two-fold by ZipA
Additional file 1: Figure S3a), whereas FtsZ wild type
and E83Q showed a fast polymerization kinetics that
could not be measured. E83Q formed polymers that
remained in solution around three times longer than
those formed by FtsZ wild type, with a rate of
depolymerization 3.9 times slower. This increase in the
time in which the filaments remain polymerized can be
explained by the small apparent catalytic constant for
the GTPase activity. The time traces of light scattering
(Additional file 1: Figure S4) indicate that the polymerization and the subsequent depolymerization of FtsZ are
altered by the mutations on the arginine 85 and glutamate 83. Additional file 1: Figure S5 shows that calcium increased the ligth scattering, and for FtsZ wild
type and E83Q this was due to the formation of bundles, as shown in Additional file 1: Figure S6. On the
other hand, R85Q presented a smaller increase in light
scattering and no formation of bundles was observed.
Figure 5A shows the maximum light scattering value
reached by FtsZ in 1 mM GTP in the presence and absence of ZipA. The mutants presented a decrease in the
polymerization levels of ~20% and ~75% for E83Q and
R85Q, respectively. In the presence of ZipA, the maximum light scattering value of FtsZ wild type increased
slightly, (~20%, and bundles were observed by electron
microscopy, Additional file 1: Figure S6), while for both
mutants the increment was significantly larger: 90% and
150% for E83Q and R85Q, respectively (Figure 5A). In the
absence of ZipA, FtsZ wild type polymerized into long
and mostly straight filaments of 8 to 24 nm width (media
around 14 nm) suggesting that the main population of
these filaments consists of 4 assembled protofilaments of
4 nm width for each protofilament (Figure 5Ba and Ca').
E83Q polymerized into curved filaments with a width of 8
to 12 nm (Figure 5Bb and Cb'). The greatest effect was
caused by the mutation R85Q, in which the polymers were
mainly short and straight, and occasionally, spirals probably composed of single or double protofilaments were
found on the grids (Figure 5Bc and Cc'). ZipA induced
bundling of wt and E83Q, which is in agreement with previous studies [34]; however, we did not observe a clear
bundling effect in the R85Q mutant, even given the increase in light scattering. The light scattering contribution
of ZipA was subtracted, therefore we can rule out the possibility that the increase in the light scattering of R85Q is
due to ZipA. Further characterization of R85Q will be
done in a future study.
All the results together indicate that arginine 85 and
glutamate 83 participate in the longitudinal and in the
lateral interactions, shown by their reduced GTPase activity and the effect on polymerization, respectively.
Interestingly, the addition of a stabilizer of the lateral
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interactions, such as ZipA to R85Q, did not induce bundles (Additional file 1: Figure S6) but stimulated the
GTPase activity (Figure 4B and Additional file 1: Figure
S3b), indicating that protofilaments are induced. On the
other hand, in the case of E83Q, even though the protofilament lateral interactions were diminished (Figure 5B)
and ZipA induced bundles that were thinner than those
with FtsZ wild type, the GTPase activity was not greatly
compromised. Therefore, we believe that the ability of
FtsZ to form bundles, presumably through lateral interactions, is important to form the Z-ring in the cell, and
consequently for cell division, a conclusion consistent
with our immunofluorescence experiments (Figure 3)
and cell viability results (Figure 2).
Discussion
The aim of this work was to characterize the role of two
conserved amino acid residues, E83 and R85, located in
the bend of helix H3 at the lateral face of FtsZ (Figure 1A).
Our results show that these amino acids are relevant for
cell division, FtsZ polymerization, GTPase activity and stability of the polymers.
The properties of these FtsZ mutants were evaluated
through their kinetics and critical concentrations for
polymerization and GTPase activities in the absence and
presence of ZipA and calcium. The FtsZ mutant proteins
had the same secondary structure as the wild type form
(within the experimental error) indicating an intact global
fold. Interestingly, although the mutations are located far
from the GTPase catalytic site (at the logitudinal interface
within the filaments) both mutant proteins hydrolyzed
GTP with less efficiency than wild type in the following
order: WT > E83Q > R85Q. Compared to the wild type
FtsZ, filaments with mutant proteins were thinner with
altered curvatures, indicating that the mutations affect
lateral interactions between FtsZ protofilaments. ZipA,
which in wild type FtsZ is known to induce bundling without affecting GTPase activity, induced thinner bundles in
E83Q, while in the case of R85Q was unable to cause
bundling, suggesting that both glutamate 83 and arginine
85 are important for lateral interactions between protofilaments. R85Q filaments were short and abnormally bent,
indicating that the mutation also affects the geometry of
the longitudinal interactions. The formation of short filaments is consistent with the ability of R85Q to hydrolyze
GTP, which requires longitudinal interaction between two
FtsZ molecules [35-38]. Also the neighbor mutation
D86K, which induces twined filaments, shows a reduction
in GTPase activity further suggesting a role of this region
in longitudinal interactions [11]. The long lag period of
the progress curve of R85Q GTPase activity was suppressed by increasing the protein concentration, confirming that this activity was directly related to polymerization.
This result could be explained by a reduction of the
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Figure 5 Polymerization of E83Q and R85Q in presence of ZipA. (A) The polymerization of the FtsZ mutants and wild type was followed by
light scattering; the buffer and protein concentrations were the same as described in Figure 4. After a stable base line of the buffer containing
1mM GTP, with or without ZipA, FtsZ was added to initiate the polymerization reaction. The change in light scattering at 350 nm was recorded
during the polymerization reaction and the difference between the base line and the maximum value reached after FtsZ addition is shown in the
bar graph. (B) Electron microscopy of negatively stained polymers of wild type FtsZ, E83Q and R85Q polymerized as in (A). The samples for FtsZ
WT, E83Q and R85Q were taken at 23 min, 53 min and 2h 50 min, respectively (see Additional file 1: Figure S4 for polymerization curves). Electron
micrographs were taken at a magnification of 28,500 for wild type FtsZ and E83Q, and of 52,000 for R85Q. Scale bar, 50 nm. (C) Histogram of FtsZ
polymers width distribution shows that the mutations E83Q and R85Q decrease the number of protofilaments per polymer. At least 31 polymers
were measured for each case using ImageJ.
Shin et al. BMC Microbiology 2013, 13:26
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nucleation free energy over the free energy for the elongation process.
The bend in H3 is likely caused by the negativelycharged amino acids DED located just before the bend
which destabilize the helix by negative charge repulsion.
At the other end of the H3 bend, R85 is followed by D86;
therefore, the positive charge of R85 could destabilize the
positive dipole of the helix in this region. Thus, altering
the charges in this region could induce a conformational
change, explaining the effects of the R85Q, E83Q and
D86K [24] mutations on the functionality of FtsZ.
Based on our results we hypothesize that, directly or via
a subtle conformational change, the charges of amino acids
located in the bend of the H3 helix are involved in longitudinal and lateral interactions. We infer that the negative
charge of glutamate 83 weakens these interactions while
the positive charge of arginine 85 stabilizes them, probably
regulating the dynamics of the Z-ring. Thus, cells carrying
FtsZ(E83Q) or FtsZ(R85Q) were affected in cell division
and showed reduced survival and cell filamentation in
agreement with previously described lateral-interactionFtsZ mutants that could not complement the temperature
sensitive ftsZ84 [10]. We have quantified survival rates at
the restrictive temperature (42°C) as a means to evaluate
the effect of FtsZ mutations on cell division, similar to
studies on the effects of proteins expression from unstable
plasmids [39,40].
The E83Q and R85Q mutations diminished the apparent catalytic constant of GTPase activity by 60% and 95%,
respectively. Presumably, the positive charge of R85 is
involved in longitudinal and lateral interactions between
protofilaments explaining the inhibitory effect of the
R85Q mutation on GTPase activity and polymerization
when compared to the E83Q or D86K mutants (Table 1)
[11]. Both, longitudinal and lateral interactions are important in models for the conversion of GTP hydrolysis into
mechanical energy during Z-ring constriction [8,25]. The
observed contraction of tubular liposomes in the presence
of GTP and a chimeric, membrane-attached FtsZ indicates that polymerization of FtsZ is sufficient to generate a
mechanical force to bend the membrane. Our results indicate that H3 bend mutations affecting longitudinal and
lateral interactions in vitro reduce cell viability in vivo
while they cause cell filamentation. Cell filamentation and
the immunofluorescence analysis (Figure 3) together with
the polymerization results support a model of a Z-ring
composed of several polymers that are recruited to the
septation site by lateral interactions, as has been found
in vivo [13].
Conclusion
This study describes the role of the positively charged arginine 85 and the negatively charged glutamate 83 in longitudinal and lateral interactions to form FtsZ protofilaments
Page 10 of 13
Table 1 Summary of FtsZ mutations and their effects on
cell viability, GTPase activity and polymer morphology
Location of the
mutation
Polymer
Viabilityb,
GTPase
%
activityc, % morphologyd
Ref.
Lateral
sidea
aa, nº
ZipA
WT
100
100
B
This study
Left
E83Q
10
40
B
This study
Left
R85Q
0.5
5
S
This study
Left
D86K
N
49
Nd
[11]
Right
E250A
N
67
Nd
[11]
a
The mutations are positioned on the surface and their location on the
structure are shown in Figure 1. Left: the left lateral surface; Right: the right
lateral surface.
aa, nº = amino acid number.
b
The % viability was determined from data of Figure 2. N = no growth of
ftsZ84 at the restrictive temperature.
c
GTPase activity was measured with 12 μM of protein in all cases.
d
B, bundles; S, short filaments; Nd, not determined. The shape of the polymers
was determined by electron microscopy (Figure 5).
and two-dimensional polymers of protofilaments, respectively. We also show that GTPase activity could be finetuned by the net charge of the H3 helix bend. Finally, our
results suggest that the conserved amino acids E83Q and
R85Q located in the bend of H3 are involved in the formation of a functional Z-ring.
Methods
Bacterial strains and plasmids
Strain C41(DE3), cured from C41/pHis17-MJ0370 [41],
was used to express E. coli FtsZ WT and mutants. VIP2
(DE3)/pLAR9 was used to evaluate the functionality of
FtsZ in vivo. VIP2(DE3)/pLAR9 is VIP2/pLAR9 [31] lysogenized with the λDE3 Lysogenization Kit (Novagen).
pMFV57 was constructed from pMFV56 [31] by cloning
the EcoRV β-lactamase fragment into the kanamycinr
(kan) gene. The β-lactamase gene was generated by PCR
using the following primers: 5′-CATTCAAATATGGAT
CCGCTCATG-3′ and 5′-ACCAATGCTTAATCAGTGA
GG-3′. FtsZ E83Q and R85Q mutants were constructed
using the QuikChange XL Site-Directed Mutagenesis Kit
(Stratagene) and the pairs of primers used to generate the
mutations were the following: 5′-GCAATGCGGCTGAT
CAGGATCGCGATGCATTGC-3′ and 5′-GCAATGCA
TCGCGATCCTGATCAGCCGCATTGC-3′; 5′-GCGGC
TGATGAGGATCAGGATGCATTGCGTGCG-3′ and 5′CGCACGCAATGCATCCTGATCCTCATCAGCCGC-3′,
respectively. The presence of the ftsZ gene mutations was
confirmed by DNA sequencing and the new plasmids were
named pMFV-E83Q and pMFV-R85Q. These constructions are derivatives of the pET28 expression system; FtsZ
expression is thus inducible by the addition of IPTG.
Shin et al. BMC Microbiology 2013, 13:26
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Protein purification
E. coli FtsZ(WT), FtsZ(E83Q) and FtsZ(R85Q) mutants
were purified as described previously [42] with some
modifications. E. coli C41(DE3) carrying the appropriate
pMFV-plasmid derivative was grown in LB-ampicilin
(amp) with shaking at 37°C. When the OD550 reached
0.5, IPTG was added to a final concentration of 0.5 mM
and the culture was grown for a further 3 h. Harvested
cells were washed and then suspended in buffer A (50 mM
Tris–HCl, pH 7.9, 50 mM KCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 10% glycerol). After lysis by sonication (Sonifier Cell Disruptor Model W185, 1 cm
diameter), the suspension was centrifuged at 28,000
rpm, at 4°C for 90 min (Beckman L5-75B Ultracentrifuge,
rotor type 30) to remove the membrane fraction and the
supernatant was collected. The proteins were precipitated
by adding 35% ammonium sulfate, centrifuged in the same
rotor at 15,000 rpm for 20 min and the pellet was dissolved
in buffer A. The dialyzed ammonium sulfate fraction was
loaded onto a Q-Sepharose column (25 cm × 4.6 mm) and
eluted with a 50 mM – 1 M gradient of KCl at a flow rate
of 1 ml/min. The FtsZ fractions were loaded onto a
Sephacryl S-400 column and eluted with buffer A at a
flow rate of 1 ml/min. The peak fractions were pooled,
concentrated to 10 – 15 mg protein/ml using Centriprep,
fast frozen and stored at −80°C. The E. coli FtsZ obtained
was pure, as determined by SDS-PAGE gels and western
blotting.
In vivo cell survival assay
To evaluate the functionality of the FtsZ(E83Q) and
FtsZ(R85Q), the viability of cells expressing these
mutants was determined using the strain E. coli VIP2
(DE3)/pLAR9 (also called VIP5). In this strain the ftsZ
chromosomal copy is interrupted by an insertion (resistant to kanamycin (kan)), and complemented with a copy
encoded on the thermosensitive plasmid pLAR9, resistant to chloramphenicol (cam) [31]. The assay was performed as described by Díaz-Espinoza et al. [39]. Briefly,
overnight cultures of VIP5 transformed with or without
pMFV-WT, pMFV-E83Q or pMFV-R85Q, were diluted
1:100 in LB media containing 100 μg/ml amp and grown
at 30°C until the OD550 reached 0.5 – 0.6. Serial dilutions of this culture plated on LB-amp were incubated,
at both the permissive (30°C) and non-permissive temperatures (42°C). The next day, the survival was determined for wtFtsZ and each of the mutants.
GTPase activity
GTPase activity was monitored by determining the area
under the peaks corresponding to GDP and GTP from
the HPLC chromatograms obtained after loading the deproteinized reaction samples onto a SUPERCOSIL LC18-DB column (SUPELCO) as previously described [43].
Page 11 of 13
FtsZ was incubated for 2 min in polymerization buffer
(50 mM Mes pH 6.5, 50 mM KCl, 10 mM MgCl2) at 30°C
and the reaction was started by addition of GTP (1 mM
final concentration). At different times, 100 μl samples
were withdrawn and transferred to 8 μl 70% perchloric
acid and neutralized by the addition of 100 μl 1.2 M
K2CO3. After degasification in a LABCONCO Speed-Vac
at room temperature for 5 min, the reaction was centrifuged at 14,000 rpm for 15 min and a 20 μl aliquot of the
supernatant was loaded onto the SUPERCOSIL LC-18-DB
column and eluted at 1 ml/min using a buffer containing
0.2 M K2HPO4, 0.1 M acetic acid and 4 mM tetrabutylammonium phosphate monobasic (TBAP) in an HPLC. GDP
and GTP were detected at 260 nm by a UV detector system. Alternatively, the inorganic phosphate product of
GTP hydrolysis was determined using the colorimetric
method of malachite green [44]. In brief, the polymerization sample (70 μL) was mixed with perchloric acid (10%
final concentration). After 5 minutes, 50 μL of this solution
were mixed in a vortex for 1 min with 800 μl malachite
green reagent 0.045%. 100 μl of citrate (34%) were added
and the solution was kept on ice for 20 min before the
measurement of the absorbance at 630 nm.
E. coli FtsZ polymerization by light scattering
Assembly of FtsZ was monitored using the Luminescence
Spectrometer LS 50 (Perkin Elmer) as described previously [33]. Excitation and emission wavelengths were set
to 350 nm, with a slit width of 5 nm. 12.5 μM FtsZ was
incubated in polymerization buffer at 30°C in a fluorescence cuvette with a 1 cm path length and after 2 min of
data collection, GTP was added to a final concentration of
1 mM.
Immunofluorescence of E. coli FtsZ and ZipA
To label FtsZ and ZipA, cells were treated following the
method described by Rueda et al. [45]. This method has
three steps: fixation, permeabilization and development
with specific antibodies. A) Fixation. In order to compare
the localization of the proteins at 30°C and 42°C, duplicated cellular cultures of VIP2/pLAR9, complemented
with FtsZ(WT) and the mutants FtsZ(E83Q) or FtsZ
(R85Q) were grown. The cultures grown at 30°C contained the antibiotics kan, amp and cam, while cultures
grown at 42°C only contained kan and amp. The negative
control was the strain VIP2/pLAR9 without pMFV57, and
no amp was added to the culture. After the cell culture
reached an OD600 of 0.5, one of the duplicated tubes was
incubated at 42°C and the growth continued for another
4 h. The cells were fixed with 0.75% formaldehyde and
incubated for 10 min at room temperature followed by
30 min on ice. The fixed cells were washed with PBS and
suspended in buffer GTE (20 mM Tris–HCl pH 7.5,
50 mM glucose and 10 mM EDTA). B) Permeabilization.
Shin et al. BMC Microbiology 2013, 13:26
http://www.biomedcentral.com/1471-2180/13/26
8 μg/ml final concentration of lysozyme were added to
30 μl of fixed cells, placed on a glass slide coated with
poly-L-lysine and incubated at room temperature for
2–3 min and washed with PBS. C) Development was carried out with specific antibodies against FtsZ and ZipA.
The unspecific binding sites were blocked by adding 3%
BSA in PBS for 15 min at room temperature and this solution was replaced by a solution containing an appropriate
dilution of the primary polyclonal antibody (anti-FtsZ or
anti-ZipA). After an overnight incubation at 4°C, the cells
were washed with PBS and incubated for 2 h with the secondary antibody (anti-rabbit labeled with Alexa 488, Invitrogen) at room temperature in darkness. After several
washes with PBS, a drop of antifading (Vectors Lab) was
added and the samples covered with a cover slide. Cells
were observed in a confocal microscope (Leica TCS SP2).
Electron microscopy
Staining and visualization of the FtsZ polymers were carried out in a JEOL JEM-100SX electron microscope. The
FtsZ polymers in absence or presence of ZipA were incubated at 30°C in polymerization buffer with 1 mM GTP.
After the maximal polymerization was reached (as detected
by light scattering), 10 μl of the polymerization reaction
was placed on a carbon-coated grid for 1 min and stained
with 1% of aqueous uranyl acetate. Electron micrographs
were taken at different magnifications. Each picture was
digitalized directly from the film and the width of the polymers was measured using the computer program ImageJ
1.34 s (http://rsb.info.nhi.gov/ij/download.htlm).
Endnotes
a
The 3D structure of the E. coli FtsZ model was built
with the program MODELLER v6.2 using as template
the crystal structures of M. jannaschii FtsZ (1FSZ.pdb)
and P. aureginosa FtsZ (1OFU.pdb). The program gave
a rms value of 0.31 Å for the structural fit. The good
quality of the model was assessed with the computer
programs PROSA II and Verify 3D (web site http://
nhiserver.mbi.ucla.edu/Verify_3D/).
Additional file
Additional file 1: Figure S1. Cellular localization and distribution of
FtsZ WT and the mutants E83Q and R85Q. Figure S2. Localization and
distribution of ZipA in cells expressing FtsZ WT and mutants E83Q and
R85Q. Figure S3. Effect of ZipA on the polymerization of FtsZ R85Q
using light scattering and GTPase activity. Figure S4. Polymerization
kinetics of FtsZ WT and the mutants E83Q and R85Q detected by light
scattering. Figure S5. Effect of Ca2+ on the polymerization of FtsZ WT
and the mutants E83Q and R85Q using light scattering. Figure S6. Effect
of Ca2+ and ZipA on the polymers morphology of FtsZ WT and the
mutants E83Q and R85Q.
Page 12 of 13
Competing interest
The authors declare no conflict of interest.
Authors’ contributions
JYS contributed to the conception and design of the study, and conducted
the experiments. OM and RL conceived the study, designed the experiments,
analyzed and interpreted the data. WV designed, conducted and supervised
the electron and confocal microscopy experiments. JYS, WV, RL and OM
wrote the manuscript. All authors have no conflicts of interest to report. All
authors read and approved the final manuscript.
Acknowledgments
We thank Drs. David Jameson and Michael Handford for the critical review of
the manuscript. Andrea Poch and Felipe Hurtado for cell morphology
experiments (not shown), Christian Escobar and Gissela Araya for
polymerization experiments (not shown). We are grateful to Dr. Debabrata
RayChaudhuri for the ZipA plasmid donation and Dr. Carlos Bustamante for
the use of his microscope facility for cell morphology studies. This work was
supported by EC FP7 Grant DIVINOCELL # 223431 and FONDECYT Grant #
1095121. The stay of J.Y. Shin in the laboratory of Dr. W. Vollmer was
financed by DAAD.
Author details
1
Department of Physics, University of California, Berkeley. 2Institute for Cell
and Molecular Biosciences, Centre for Bacterial Cell Biology, Newcastle
University, Richardson Road, Newcastle upon Tyne NE2 4AX, UK.
3
Departamento de Biología, Facultad de Ciencias, Casilla 653, Santiago, Chile.
Received: 14 May 2012 Accepted: 25 January 2013
Published: 5 February 2013
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doi:10.1186/1471-2180-13-26
Cite this article as: Shin et al.: Glutamate 83 and arginine 85 of helix H3
bend are key residues for FtsZ polymerization, GTPase activity and
cellular viability of Escherichia coli: lateral mutations affect FtsZ
polymerization and E. coli viability. BMC Microbiology 2013 13:26.
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